1. Field of the Invention
The present invention relates to expression cassettes for transforming Pichia ciferrii. More particularly, it relates to expression cassettes containing Pichia ciferrii ribosomal DNA fragment, CYHr gene resistant to cycloheximide, and a desired gene, and to uses thereof.
2. Description of the Prior Art
Pichia ciferrii has been used to biologically desulfurize coals (Stevens et al., U.S. Pat. No. 4,851,350), to produce D-alpha-amino acids (Takelchi et al., U.S. Pat. No. 5,068,187), to produce (S)-1-phenyl-1,3-propandiol (Ajinomoto, JP 6-90789A) or to produce secondary alcohols by stereospecific ketone reduction (Merck, EP-300287). Further, it produces and secretes tetraacetyl phytosphingosine (TAPS) which is a precursor of ceramides (Barenholz et al., Biochem. Biophys. Acta, 248, 458, 1971; ibid, 306, 341, 1973).
Phytosphingosines including TAPS, like ceramides, show an activity of surface skin-protection and of preventing excessive water-loss and dry out of the skin, facilitating their uses in cosmetics. They can be obtained from various microorganisms and easily converted to ceramides by N-acylation.
TAPS productions by wild type Pichia ciferrii ATCC 14091 and F-60-10 (NRRL 1301) are not satisfactory for commercial uses. To improve the production of TAPS in the strains of Pichia ciferrii, attempts to provide mutants which are capable of producing a higher level of TAPS have been made (Wickerham and Burton, J. Bacteriol., 80, 484, 1960; U.S. Pat. No. 5,618,706). The present inventors also developed novel useful mutant (KFCC-10937) which allows a larger amount of TAPS production in a shorter time (KR 98-49305A).
Pichia ciferrii had been classified into genus Hansenula and is recently reclassified into genus Pichia by 5S-RNA analysis (Yamada et al., Biosci, Biotechnol. Biochem., 58, 1245, 1994). By this reason, the genetic study of the Pichia yeasts is not sufficient and transformation method of Pichia ciferrii has not been established.
The present invention provides plasmid prACL2 comprising Pichia ciferrii serine palmitoyl transferase gene and a transformed Pichia ciferrii cell which allows an improved production of TAPS.
The inventors found that the known transformation method for Candida utilis (Kondo et al., J. Bacteriol., 177, 7171, 1995) can be modified and applied to the Pichia ciferrii. They cloned Pichia ciferrii ribosomal protein L41-coding gene to determine its nucleotide sequence and manipulated to give a resistance to cycloheximide, an antibiotic from yeasts, so as to be used as a selection marker. Thus recombinant gene may be linked to a plasmid which carry a desired gene to give an expression cassette which is useful to transform Pichia ciferrii in which the desired gene is expressed.
Moreover, the inventors succeeded in cloning of Pichia ciferrii GAPDH promoter gene and found that its insertion into the expression cassette allows an unexpected improvement of the expression level. In fact, they increased the production amount of TAPS by transforming the strain of Pichia ciferrii with the expression cassette carrying LCB2 gene as a desired gene and culturing the resulting transformed cells. LCB2 gene codes for palmitoyl transferase which is involved in the TAPS synthesis in the living body.
Accordingly, an object of the present invention is to determine and use genetic information of Pichia ciferrii ribosomal protein L41 gene.
Another object of the present invention is to provide an expression cassette for transforming Pichia ciferrii, which comprises Pichia ciferrii ribosomal DNA, Pichia ciferrii L41 gene as a selection marker, and a desired gene. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.
Another object of the present invention is to provide a method for transforming Pichia ciferrii with a plasmid containing the expression cassette.
The present invention determines and uses genetic information of Pichia ciferrii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and GAPDH promoter gene.
The present invention provides an expression cassette for transforming Pichia ciferrii, which comprises Pichia ciferrii ribosomal DNA, Pichia ciferrii L41 gene as a selection marker, Pichia ciferrii GAPDH promoter gene and a desired gene. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.
The present invention further provides an expression cassette for transforming Pichia ciferrii, which comprises Pichia ciferrii ribosomal DNA, Pichia ciferrii L41 gene as a selection marker, Pichia ciferrii GAPDH promoter gene, a desired gene and Pichia ciferrii ribosomal DNA. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.
The present invention determines and uses genetic information of Pichia ciferrii serine palmitoyl transferase which is involved in TAPS synthesis.
The present invention provides plasmid prACL2 comprising an expression cassette having Pichia ciferrii serine palmitoyl transferase gene and a transformed Pichia ciferrii cell with an improved production of TAPS.
The present invention further provides plasmid prACGL2 comprising an expression cassette having Pichia ciferrii serine palmitoyl transferase gene and a Pichia ciferrii transformant with an improved production of TAPS.
The present invention further provides plasmid prHECGL2 comprising an expression cassette having Pichia ciferrii serine palmitoyl transferase gene and a Pichia ciferrii transformant with an improved production of TAPS.
The present invention still further provides a method for producing TAPS by culturing the transformed Pichia ciferrii cells.
The objects mentioned above, other features and applications of the present invention would be much more apparent by those of ordinary skills in the art from the following explanation in detail.